The development of nucleic acid amplification technology has revolutionized genetic analysis and engineering science. For example, the polymerase chain reaction (PCR) is commonly utilized to amplify specific target nucleic acids using selected primer nucleic acids, e.g., to facilitate the detection of target nucleic acid as part of a diagnostic, forensic or other application. Primers typically function in pairs that are designed for extension towards each other to cover the selected target region. A typical PCR cycle includes a high temperature (e.g., 85° C. or more) denaturation step during which the strands of double-stranded nucleic acids separate from one another, a low temperature (e.g., 45-65° C.) annealing step during which the primers hybridize to the separated single strand, and an intermediate temperature (e.g., around 72° C.) extension step during which a nucleic acid polymerase extends the primers. Two-temperature thermocycling procedures are also utilized. These generally include a high temperature denaturation step and a low temperature anneal-extend step.
Various strategies for detecting amplification products have been developed and one of the most widely used method is the 5′ nuclease or TaqMan® assay. The 5′ nuclease assay typically utilizes the 5′ to 3′ nuclease activity of certain DNA polymerases to cleave 5′ nuclease oligonucleotide probes during the course of PCR. This assay allows for both the amplification of a target and the release of labels for detection, generally without resort to multiple handling steps of amplified products. The 5′ nuclease probes typically include labeling moieties, such as a fluorescent reporter dye and a quencher dye. When the probe is intact, the proximity of the reporter dye to the quencher dye generally results in the suppression of the reporter fluorescence. During a 5′ nuclease reaction, cleavage of the probe separates the reporter dye and the quencher dye from one another, resulting in a detectable increase in fluorescence from the reporter. The accumulation of PCR products or amplicons is typically detected indirectly by monitoring this increase in fluorescence in real time.
Many reagents are required to perform a PCR assay and reagents such as DNA polymerase, deoxyribonucleoside triphosphates (dNTPs), oligonucleotide primers, probes, and salts (magnesium, potassium, chloride) in pH-maintaining buffers (e.g. Tris-HCl) are often pre-mixed in solutions referred as mastermixes. It has been known also that certain materials such as gelatin, bovine serum albumin (BSA), ammonium sulfate, and nonionic detergents act as stabilizing agents and improve the performance of a PCR assay. The addition of glycerol (15-20%) to a PCR mixture can also enhance PCR reaction performance by increasing the thermal stability of DNA polymerase and also by lowering the temperature necessary for strand separation (see Cheng, S. et al., Proc. Natl. Acad. Sci. USA, 91, 5695, 1994).